CN105886510B - The application of long-chain non-coding RNA ENST00000529841 - Google Patents
The application of long-chain non-coding RNA ENST00000529841 Download PDFInfo
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- CN105886510B CN105886510B CN201610436542.2A CN201610436542A CN105886510B CN 105886510 B CN105886510 B CN 105886510B CN 201610436542 A CN201610436542 A CN 201610436542A CN 105886510 B CN105886510 B CN 105886510B
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Abstract
The invention discloses a kind of long-chain non-coding RNA (long no coding RNA, LncRNA) the application of ENST00000529841, the mescenchymal stem cell in prediction people's umbilical cord source is used to prepare to the detection reagent of osteoblast differentiation, especially prepares the mescenchymal stem cell in Real time PCR method prediction people's umbilical cord source to the kit of osteoblast differentiation.Confirm LncRNA ENST00000529841 after osteogenic induction culture processing by research, ENST00000529841 high is expressed, therefore, the expression of ENST00000529841 is used for prediction of the mescenchymal stem cell to osteoblast differentiation, there is far-reaching clinical meaning and generalization.
Description
Technical field
The present invention relates to stem cell molecular biology fields, and in particular to long-chain non-coding RNA ENST00000529841
Preparing application of the mescenchymal stem cell in prediction people's umbilical cord source into osteoblast differentiation preparation.
Background technology
Mescenchymal stem cell (Mesenchymal stem cells, MSCs) is a kind of with the of self-replication capacity and multidirectional
The adult stem cell of differentiation potential belongs to non-terminally differentiated cells, in vitro under specific inductive condition, can be divided into fat,
The Various Tissues cell such as cartilage, bone, muscle, tendon, nerve, liver, cardiac muscle, beta Cell of islet and endothelium, continuous passage culture and cold
Freezing still has multi-lineage potential after preserving, be a kind of ideal Seeding Cells in Bone Tissue Engineering.Current most widely used bone
The mescenchymal stem cell in the sources such as marrow, fat, umbilical cord and Cord blood, they all have multi-lineage potential；And it is easily obtained
And amplification, still there is multi-lineage potential after continuous passage culture and freezen protective, can be that the reparation of histoorgan and regeneration carry
For required a large amount of cells；Immunogenicity is low simultaneously, whether self or allogenic mescenchymal stem cell, generally not
It can cause the immune response of host, also there is unique immunoloregulation function.
Long-chain non-coding RNA (Long non-coding RNA, lncRNAs) is that one kind does not encode egg more than 200bp
White RNA.With universal and to LncRNAs the understanding of high throughput sequencing technologies, it has been found that LncRNAs is in gene expression
Very important effect is played in regulation and control.Equally during mescenchymal stem cell directed differentiation, LncRNA is also by regulation and control
The expression of related gene affects the direction of differentiation.
Invention content
The object of the present invention is to provide a kind of applications of LncRNA ENST00000529841.LncRNA
ENST00000529841(NONCODE ID：NONHSAT024154.2 No. 11 chromosomes, expression energy conduct) are positioned at
Judge whether umbilical cord mesenchymal stem cells are induced to differentiate into the foundation of osteoblast, therefore can be used for preparing prediction people's umbilical cord and come
The mescenchymal stem cell in source to the preparation of osteoblast differentiation, be further capable of providing it is a kind of cost-effective, it is easy to spread to answer
The kit of skeletonization efficiency evaluation.
The application of long-chain non-coding RNA ENST00000529841, the mesenchyma for being used to prepare prediction people's umbilical cord source are dry
Cell is to osteoblast differentiation preparation, the transcript sequence such as SEQ NO of long-chain non-coding RNA ENST00000529841：1
It is shown.
The mescenchymal stem cell in prediction people's umbilical cord source includes detecting the non-volume of long-chain to osteoblast differentiation preparation
The real-time fluorescence quantitative PCR detection reagent of code RNA ENST00000529841 expression quantity.
The real-time fluorescence quantitative PCR detection reagent includes the specific primer of real-time fluorescence quantitative PCR：lncRNA
ENST00000529841 specific forward primers：5'-ACTACTTGGCCTGCTCAGTG-3'lncRNA
ENST00000529841 specific reverse primers：5'-GCATAGCAAAACCCCATCTC-3'.
To the kit of osteoblast differentiation, which includes for a kind of mescenchymal stem cell in prediction people's umbilical cord source：
LncRNA ENST00000529841 specific forward primers：5'-ACTACTTGGCCTGCTCAGTG-3'
LncRNA ENST00000529841 specific reverse primers：5'-GCATAGCAAAACCCCATCTC-3'.
The kit further includes：
The specific primer of internal reference β-actin：
Forward primer 5'-CCTATCGAGCATGGAGTGGT-3'
Reverse primer 5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin：
Forward primer 5'-CCTGATGTATGCCAAACGTG-3'
Reverse primer 5'-TCCCTGTAAAAGCAGCACCT-3'.
The kit full set reagent includes：
(1) from the mescenchymal stem cell extracted total RNA agents useful for same of induction, including Trizol reagents, chloroform, isopropyl
Alcohol, no enzyme water；(2) it is cDNA agents useful for same by lncRNA ENST00000529841 reverse transcriptions by template of total serum IgE, including inverse
Transcription buffer, dNTP, RNase inhibitor, MMLV reverse transcriptases and random primer；It (3) will be used in cDNA real-time quantitative PCRs
Reagent, including the specific primer of lncRNA ENST00000529841, real time fluorescent quantitative SYBR dyestuffs, without enzyme water.
The present invention induces the mescenchymal stem cell in people's umbilical cord source by Osteogenic Induction Medium, after handling 1,2,3 weeks, carries
Cell total rna is taken, the expression of real-time fluorescence quantitative PCR analysis LncRNA ENST00000529841 is carried out after reverse transcription, is found：
Induction LncRNA ENST00000529841 expression in 14 days is 25 times or so not induced, expresses and increases significantly after induction, other
Classical osteoblast molecule marker such as osteocalcin and osteopontin also increases, but multiple is not so good as LncRNA
The expression of ENST00000529841 is increased significantly.Accordingly, applicant proposes to prepare using LncRNA ENST00000529841 pre-
The mescenchymal stem cell in people's umbilical cord source is surveyed to osteoblast differentiation reagent.
By LncRNA ENST00000529841 for predicting whether the mescenchymal stem cell in people's umbilical cord source is thin to skeletonization
The method of born of the same parents' differentiation：(1) mescenchymal stem cell in people's umbilical cord source not induced after osteogenic induction or, extracted total RNA are collected；
(2) it is cDNA by LncRNA ENST00000529841 reverse transcriptions by template of total serum IgE；(3) LncRNA is used
ENST00000529841 specific primers and internal control primer carry out real-time fluorescence quantitative PCR amplification and obtain relative expression quantity, induction
The expression difference not induced is apparent, can as whether the prediction reagent of Osteoblast Differentiation.
Using the present invention reagent can detect people umbilical cords of the LncRNA ENST00000529841 after osteogenic induction come
The expression of the mescenchymal stem cell in source carries to judge whether Osteoblast Differentiation for human umbilical cord mesenchymal stem cells Osteoblast Differentiation
The molecular marker of lncRNA has been supplied, there is far-reaching practice significance and generalization.
It further illustrates the present invention, is not intended to limit the present invention below in conjunction with description of the drawings and specific implementation mode.
Description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR is analyzed after LncRNA ENST00000529841 osteogenic inductions or the people that does not induce
The differential expression of the mescenchymal stem cell in umbilical cord source；Although the expression of induction 21 days will be less than induction 14 days, it may be possible to
Caused by other genes or the factor jointly regulation and control, no matter but the length of induction time will compare after capable of still finding out induction
Expression before induction obviously increases；The lncRNA ENST00000529841 can be used as umbilical cord mesenchymal stem cells to skeletonization
Cell differentiation marker；
QC1205con representatives ordinary culture medium culture group；
QC1205diff representatives Osteogenic Induction Medium culture group.
Specific implementation mode
1 Osteogenic Induction Medium of embodiment induce 1,2,3 week and the human umbilical cord mesenchymal stem cells that do not induce in LncRNA
The detection kit of ENST00000529841
1. isopropanol 100ml
2.Trizol reagents 100ml
3. chloroform 50ml
4. 1 μM of random 50 μ l of reverse transcriptase primer
5. without enzyme water 2ml
6. 10mM dNTP 100μl
7. 50 μ l of 200U/ μ l RNA reverse transcriptases
8. 5 × RT Buffer 1ml
9. 500 μ l of 40U/ μ l RNA inhibitor
10.Premix Ex Taq 50μl
11. 10 μM of 50 μ l of lncRNA ENST00000529841 specific primers
The specific primer of quantitative fluorescent PCR：
LncRNA ENST00000529841 forward primers：5'-ACTACTTGGCCTGCTCAGTG-3'
LncRNA ENST00000529841 reverse primers：5'-GCATAGCAAAACCCCATCTC-3'
12. 10 μM of each 50 μ l of internal reference control primer
The specific primer of internal reference β-actin：
5'-CCTATCGAGCATGGAGTGGT-3'
5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin：
5'-CCTGATGTATGCCAAACGTG-3'
5'-TCCCTGTAAAAGCAGCACCT-3'
The detection of LncRNA ENST00000529841 after 2 human umbilical cord mesenchymal stem cells osteogenic induction of embodiment
(1) mescenchymal stem cell in people's umbilical cord source that 1,2,3 Zhou Houhe of osteogenic induction is not induced, extracted total RNA are collected：
Culture medium in the culture plate after most induction is removed, 1ml Trizol are added, 5min is horizontally arranged at room temperature, lysate is made uniformly to divide
It is distributed in cell surface, then makes cell detachment with liquid-transfering gun piping and druming cell；Cell pyrolysis liquid is transferred in centrifuge tube, is added
200 μ l chloroforms, cover tightly centrifuge tube pipe lid, up and down oscillation 15 seconds, are stored at room temperature 3 minutes, 12,000g, 4 DEG C centrifuge 15 points
Clock.Centrifuge tube is taken out, sample is divided into three layers：Lower layer's organic phase of the supernatant water phase of light color, intermediate white layer and pink.It is small
The heart draws light color supernatant water and mutually moves to another centrifuge tube, isometric isopropanol is added, gently mixing, is stored at room temperature 10 minutes,
Then it centrifuges 10 minutes for 12,000g, 4 DEG C, in the visible RNA precipitate of tube bottom.Careful removal supernatant, is slowly added along tube wall
1mL75% ethyl alcohol, gently mixing.12,000g, 4 DEG C centrifuge 10 minutes, carefully exhaust supernatant.Drying at room temperature precipitates 2~5 minutes,
Be added 30~50 μ L without RNase water dissolution RNA precipitates, spectrophotometer detects the concentration and quality of RNA, OD260/280 ratios
Value is between 1.8-2.0, -70 DEG C of preservations.
(2) it is cDNA by LncRNA ENST00000529841 reverse transcriptions by template of total serum IgE；
Oligo(dT)18primer(100μM) 1μl
Total RNA 1μg
Without 12 μ l of enzyme water Total
Reverse transcription first step condition:62 DEG C 5 minutes
5 × RT Buffer, 4 μ l
dNTP(10mM) 2μl
1 μ l of RNase inhibitor (40U/ μ l)
1 μ l of MMLV reverse transcriptases (200U/ μ l)
12 μ l of first step product
Total 20μl
Reverse transcription second step program, 42 DEG C 60 minutes, 72 DEG C 5 minutes.
(3) real-time fluorescence quantitative PCR is carried out with LncRNA ENST00000529841 specific primers and internal control primer：
ENST00000529841 specific primers DNA sequence dna is synthesized by Invitrogen companies.
Reverse transcription product is first diluted 5 times, mixing, 20 μ l reaction systems are as follows：
SYBR Premix 2×10μl
1 μ l of ENST00000529841 or internal control primer
0.5 μ l of cDNA products
Without 20 μ l of enzyme water Total
Real-time fluorescence quantitative PCR react 95 DEG C 30 seconds, 40 cycle 95 DEG C 5 seconds, 65 DEG C 30 seconds, 72 DEG C 30 seconds, 65~
95 DEG C, increase by 0.5 DEG C within every 0.05 second.
LncRNA ENST00000529841 specific forward primers：5'-ACTACTTGGCCTGCTCAGTG-3'
LncRNA ENST00000529841 specific reverse primers：5'-GCATAGCAAAACCCCATCTC-3'
The specific primer of internal reference β-actin：
Forward primer 5'-CCTATCGAGCATGGAGTGGT-3'
Reverse primer 5'-CTGAGGCATAGAGGGACAGC-3'
The specific primer of internal reference α-Tubulin：
Forward primer 5'-CCTGATGTATGCCAAACGTG-3'
Reverse primer 5'-TCCCTGTAAAAGCAGCACCT-3'
(4) measurement of osteogenic induction：This experimental data uses the analysis method of relative quantification, β-actin and α-Tubulin
As reference gene, data are analyzed using GraphPad Prism.Success inducing umbilical cord mesenchymal stem is thin to skeletonization
LncRNA ENST00000529841 are significantly raised after born of the same parents' differentiation, and difference has conspicuousness (p<0.05).
Above studies have shown that lncRNA ENST00000529841 can be used as umbilical cord mesenchymal stem cells to osteoblast point
Change marker.
Claims (3)
1. the application of long-chain non-coding RNA ENST00000529841, which is characterized in that be used to prepare prediction people's umbilical cord source
Mescenchymal stem cell is to the preparation of osteoblast differentiation, the transcript sequence of long-chain non-coding RNA ENST00000529841
Such as SEQ NO：Shown in 1.
2. application according to claim 1, which is characterized in that the mescenchymal stem cell in prediction people's umbilical cord source is thin to skeletonization
The preparation of born of the same parents' differentiation includes the real-time fluorescence quantitative PCR detection for detecting long-chain non-coding RNA ENST00000529841 expression quantity
Reagent.
3. application according to claim 2, which is characterized in that the real-time fluorescence quantitative PCR detection reagent includes real
When quantitative fluorescent PCR specific primer：
LncRNA ENST00000529841 specific forward primers：5'-ACTACTTGGCCTGCTCAGTG-3'
LncRNA ENST00000529841 specific reverse primers：5'-GCATAGCAAAACCCCATCTC-3'.
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| CN103146693A (en) * | 2013-02-26 | 2013-06-12 | 中南大学 | Long chain non-coding RNA (Ribonucleic Acid) gene and application method thereof |
| CN104726570A (en) * | 2015-03-06 | 2015-06-24 | 中国人民解放军第二军医大学 | Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis |
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| CN103146693A (en) * | 2013-02-26 | 2013-06-12 | 中南大学 | Long chain non-coding RNA (Ribonucleic Acid) gene and application method thereof |
| CN104726570A (en) * | 2015-03-06 | 2015-06-24 | 中国人民解放军第二军医大学 | Kit for detecting IncRNA-NEAT1 in serum and application thereof in liver cancer serological diagnosis |
Non-Patent Citations (4)
| Title |
|---|
| detail information of NONHSAT024154.2;illumina测序数据;《Noncode数据库》;20110524;第787-798页 * |
| Long noncoding RNAs expression signatures in chondrogenic differentiation of human bone marrow mesenchymal stem cells;Wang L等;《Biochemical and biophysical research communications》;20141204;第456卷(第1期);第459-464页 * |
| MiR-29c regulates the expression of miR-34c and miR-449a by targeting DNA methyltransferase 3a and 3b in nasopharyngeal carcinoma;NIU M等;《BMC cancer》;20160315;第16卷;第1-11页 * |
| 赵培庆等.间充质干细胞成骨分化过程中长链非编码RNA调控作用研究进展.《中国医药导报》.2015,第12卷(第18期),第35-38页. * |
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